Western blot chemiluminescence reagent

A total of 8 × 10⁷ Jurkat T cells treated with siRab37 or expressing EV, Rab37-WT, Rab37-Q89L or Rab37-T43N were centrifuged at 100–300×g for 5 min at 4 °C and supernatant was discarded. The membrane fractionation was performed according to the manufacturer’s instructions (#ab65400 from Abcam, Cambridge, UK). The pellet is the membrane fraction (plasma membrane proteins), which was dissolved in 0.5% Triton X-100 in PBS and further analyzed by Western blotting. For immunoblotting, the protein was identified by incubating the membrane with primary antibodies, followed by horse radish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized using Western blot chemiluminescence reagent (Millipore, Burlington, Massachusetts, United States) and the signals were detected by Luminescence Readers (FUJI LAS-1000, Fujioka, Japan).

Cells were scraped off dishes and lysed in NET buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS. Isolated proteins were separated by SDS-PAGE and blotted onto PVDF membranes. Expression of the molecules listed below was analyzed by incubation of membrane with the indicated primary antibody at 4 °C overnight, followed by secondary antibody. ABC transporters: anti-ABCC1 (1:1000; #72202, Cell Signaling Technology; Danvers, MA, USA [CST]). USP22: anti-USP22 (1:1000; ab195289, Abcam; Cambridge, MA, USA). USPs: USP antibody sampler kit (1:1000; #12894, CST). SIRT1: anti-SIRT1 (1:1000; #9475, CST). Akt: anti-phospho-Akt and anti-Akt (1:1000; #2965/#4060/#4691, CST). Erk: anti-phospho-Erk and anti-Erk (1:1000; #4370/#4695, CST). As reference protein: heat shock cognate protein (Hsc70): mAb HSC70 (B-6) (1:5000; Santa Cruz Biotechnology; Dallas, TX, USA). Signals were developed using western blot chemiluminescence reagent (Millipore Corp.; Burlington, MA, USA) and detected with X-ray film.